Nudix enzymes, phosphoanhydrases that catalyze the hydrolysis of a diphosphate linkage in nucleotide derivative, are characterized by the presence of the signature sequence GX5EX7REUXEEX2U. Present in species ranging from prokaryotes to eukaryotes, their postulated role is to control the cellular levels of toxic metabolites and signaling molecules. The Nudix family of hydrolases can be divided into families based on their substrate specificity, catalytic activity and phenotype. The major subfamilies are: a) the MutT family, which prevents mutations by eliminating the incorporation of 8-oxo-dGTP into DNA; b) the ADP-ribose pyrophosphatases, which have been associated with tellurite resistance in bacteria; c) the ApnA hydrolases, of which an Ap5A hydrolase is an invasiveness determinant of E. coil K1; and d) the Coenzyme A pyrophosphohydrolases and NADH hydrolases for which no phenotype has yet been determined. The Nudix signature sequence, which folds as a loop-helix-loop tailored for pyrophosphate hydrolysis, contributes the catalytic center, while residues conferring substrate specificity occur in regions of the sequence removed from the Nudix motif. This segregation of catalytic and recognition roles provides versatility to the Nudix super family of hydrolases. We propose to 1) identify differences in key residues between mammalian and (myco) bacterial ADPRases that will guide the development of novel antibacterial agents; 2) identify the determinants of substrate specificity and catalytic activity of Coenzyme A pyrophosphatases, a newly identified subfamily of Nudix enzymes; and 3) determine the mechanism and the specificity of E. coil protein Ygdp, a Nudix hydrolase necessary for the invasion competence of E. coil K1. To address these questions, we designed specific experiments that will use kinetic measurements, substrate analog design, mechanistic studies, mutational studies and x-ray crystallography. The proposed research will provide insight into the details of each family of enzymes as well as basic information in the rapidly expanding family of Nudix enzymes.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM066895-04
Application #
7060040
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
Jones, Warren
Project Start
2003-05-01
Project End
2008-04-30
Budget Start
2006-05-01
Budget End
2008-04-30
Support Year
4
Fiscal Year
2006
Total Cost
$280,998
Indirect Cost
Name
Johns Hopkins University
Department
Physiology
Type
Schools of Medicine
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21218
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